Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China.
Prenatal Diagnostic Center, Guangzhou Women and Children's Medical Center, Guangzhou, Guangdong, China.
Thromb Haemost. 2023 Dec;123(12):1151-1164. doi: 10.1055/a-2107-0702. Epub 2023 Jun 7.
Hemophilia A (HA) is the most frequently occurring X-linked bleeding disorder caused by heterogeneous variants in the F8 gene, one of the largest genes known. Conventional molecular analysis of F8 requires a combination of assays, usually including long-range polymerase chain reaction (LR-PCR) or inverse-PCR for inversions, Sanger sequencing or next-generation sequencing for single-nucleotide variants (SNVs) and indels, and multiplex ligation-dependent probe amplification for large deletions or duplications.
This study aimed to develop a LR-PCR and long-read sequencing-based assay termed comprehensive analysis of hemophilia A (CAHEA) for full characterization of F8 variants. The performance of CAHEA was evaluated in 272 samples from 131 HA pedigrees with a wide spectrum of F8 variants by comparing to conventional molecular assays.
CAHEA identified F8 variants in all the 131 pedigrees, including 35 intron 22-related gene rearrangements, 3 intron 1 inversion (Inv1), 85 SNVs and indels, 1 large insertion, and 7 large deletions. The accuracy of CAHEA was also confirmed in another set of 14 HA pedigrees. Compared with the conventional methods combined altogether, CAHEA assay demonstrated 100% sensitivity and specificity for identifying various types of F8 variants and had the advantages of directly determining the break regions/points of large inversions, insertions, and deletions, which enabled analyzing the mechanisms of recombination at the junction sites and pathogenicity of the variants.
CAHEA represents a comprehensive assay toward full characterization of F8 variants including intron 22 and intron 1 inversions, SNVs/indels, and large insertions and deletions, greatly improving the genetic screening and diagnosis for HA.
血友病 A（HA）是最常见的 X 连锁出血性疾病，由 F8 基因的多种变异引起，F8 基因是已知的最大基因之一。F8 的常规分子分析需要组合多种检测方法，通常包括长距离聚合酶链反应（LR-PCR）或反转录-PCR 用于反转，Sanger 测序或下一代测序用于单核苷酸变异（SNVs）和插入缺失，以及多重连接依赖性探针扩增用于大片段缺失或重复。
本研究旨在开发一种基于 LR-PCR 和长读测序的检测方法，称为血友病 A 综合分析（CAHEA），用于 F8 变异的全面特征分析。通过与传统分子检测方法比较，在 131 个 HA 家系的 272 个样本中评估 CAHEA 的性能，这些样本具有广泛的 F8 变异谱。
CAHEA 在所有 131 个家系中鉴定了 F8 变异，包括 35 个内含子 22 相关基因重排、3 个内含子 1 反转（Inv1）、85 个 SNVs 和插入缺失、1 个大片段插入和 7 个大片段缺失。CAHEA 在另一组 14 个 HA 家系中的准确性也得到了验证。与组合使用的传统方法相比，CAHEA 检测方法在识别各种类型的 F8 变异方面具有 100%的灵敏度和特异性，并且具有直接确定大片段反转、插入和缺失的断点/点的优势，这使得能够分析重组在连接处的机制和变异的致病性。
CAHEA 代表了一种全面的 F8 变异特征分析方法，包括内含子 22 和内含子 1 反转、SNVs/插入缺失和大片段插入缺失，极大地提高了 HA 的遗传筛查和诊断。
